Compositions comprising an active agent

ABSTRACT

The present invention provides a highly dispersible formulation comprising an active agent and a dipeptide or tripeptide comprising at least two leucyl residues. The composition of the invention possesses superior aerosol properties and is thus preferred for aerosolized administration to the lung. Also provided are a method for (i) increasing the dispersibility of an active-agent containing formulation for administration to the lung, and (ii) delivery of the composition to the lungs of a subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/548,759, filed Apr. 13, 2000, now U.S. Pat. No. 6,518,239 whichclaims the benefit of priority of the following U.S. Provisional PatentApplications: Ser. No. 60/162,451 filed on Oct. 29, 1999; Ser. No.60/164,236 filed on Nov. 8, 1999; Ser. No. 60/172,769 filed on Dec. 20,1999; Ser. No. 60/178,383 filed on Jan. 27, 2000; and Ser. No.60/178,415 filed on Jan. 27, 2000, all of which are incorporated hereinby reference in their entirety.

FIELD OF THE INVENTION

The present invention is directed to highly dispersive dry powdercompositions, and in particular, to highly dispersive, inhalable drypowder compositions for aerosolized delivery to the lungs. The drypowders of the invention contain an active agent and a di- or tripeptidecontaining at least 2 leucyl residues, and are physically and chemicallystable upon storage. The powders of the invention also demonstratesuperior aerosol performance.

BACKGROUND OF THE INVENTION

Traditionally, inhalation therapy has played a relatively minor role inthe administration of biotherapeutics and conventional pharmaceuticalswhen compared to more traditional drug administration routes, such asoral and intraveneous. Injection is the customary route of delivery ofbiotherapeutics (e.g., peptides, proteins and nucleic acids), and due tothe many drawbacks associated with injection (e.g., inconvenience,discomfort, patient aversion to needle-based delivery methods),alternative administration routes are needed.

Pulmonary delivery is one such alternative administration route whichcan offer several advantages over subcutaneous administration. Theseadvantages include the convenience of patient self-administration, thepotential for reduced drug side-effects, ease of delivery by inhalation,the elimination of needles, and the like. Many preclinical and clinicalstudies with inhaled proteins, peptides, DNA and small molecules havedemonstrated that efficacy can be achieved both within the lungs andsystemically. However, despite such results, the role of inhalationtherapy in the health care field has not grown as expected over recentyears, in part due to a set of problems unique to the development ofinhalable drug formulations. Dry powder formulations, while offeringunique advantages over cumbersome liquid dosage forms andpropellant-driven formulations, are prone to aggregation and lowflowability phenomena which considerably diminish the efficiency of drypowder-based inhalation therapies.

Particulate aggregation, caused by particle-particle interactions, suchas hydrophobic, electrostatic, and capillary interactions, must beminimized in order to provide dispersible powders for effectiveinhalation therapies. Various approaches have been utilized in effortsto prepare dry powders having minimal particle aggregation and goodaerosol properties. These approaches include the modification of drypowder particle surface texture (Ganderton, et al., U.S. Pat. No.5,376,386), the co-delivery of large carrier particles (absent drug)with therapeutic aerosols to achieve efficient aerosolization, particlecoatings (Hanes, U.S. Pat. No. 5,855,913; Ruel, et al., U.S. Pat. No.5,663,198) aerodynamically light particles (Edwards, et al., U.S. Pat.No. 5,985,309), use of antistatic agents, (Simpkin, et al., U.S. Pat.No. 5,908,639) and the addition of certain excipients, e.g., surfactants(Hanes U.S. Pat. No. 5,855,913; Edwards, U.S. Pat. No. 5,985,309).Unfortunately, the formation of particulate aggregates and production ofpowders having poor flow properties and low dispersivities continue toplague development efforts to prepare aerosolizable dry powders forinhalation therapy. Thus, a need exists for improved inhalable aerosolsfor the pulmonary delivery of therapeutic agents, and in particular, fordry powders having excellent aerosol properties and reducedparticle-particle interactions, irrespective of the therapeutic agent.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery of a particular classof excipients, which, when incorporated into dry powder formulations foraerosolization and delivery to the lung, notably improves thedispersivity and aerosolization properties of the dry powders,irrespective of the type of active agent contained in the formulation.More particularly, the invention provides a dry powder composition whichcomprises an active agent and a di or tri-peptide comprising at leasttwo leucines. Preferred di- and tripeptides are those which are surfaceactive.

The dry powder of the invention typically contains from about 2% byweight to about 99% by weight di- or tri-peptide, and may optionallycontain additional excipients or carriers, such as carbohydrates, aminoacids, peptides, proteins, organic acid salts, and/or polymers.

The presence of the di- or tri-peptide is effective to notably increasethe emitted dose of the dry powder over the emitted dose of the powdercomposition absent the di- or tri-peptide. In one particular embodimentof the invention, the dry powder of the invention is characterized by anemitted dose of at least about 30%. In another embodiment, theconcentration of the dileucyl- di- or tri-peptide on the surface of theparticles is greater than in the bulk powder.

Additional features of the dry powder particles of the inventioninclude, in one embodiment, a mass median diameter of less than about 10microns, and in yet another embodiment, a mass median aerodynamicdiameter of less than about 10 microns. In yet another embodiment, thedry powder comprises particles having a bulk density from 0.1 to 10grams per cubic centimeter.

The dry powder of the invention is further characterized by bothphysical and chemical stability upon storage, as characterized, in oneembodiment, by a drop in emitted dose of no more than about 10% whenstored under ambient conditions for a period of three months. In anotherembodiment, the chemical stability of the dry powder is characterized bydegradation of less than about 5% by weight of the active agent uponstorage of the dry powdered composition under ambient conditions for aperiod of three months.

In another aspect, the invention provides a method for enhancing theaerosol performance of a dry powder. In the method, a di- or tri-peptideis incorporated into an active-agent containing liquid formulation. Theresulting liquid formulation is dried to produce a dry powder containingthe active agent and the di- and/or tripeptide, whereby the resultantdry powder possesses an emitted dose that is increased over the emitteddose of a dry powder having the same components but absent the di- ortripeptide.

In one embodiment of the method, the liquid formulation is an aqueousformulation. In another particular embodiment of the method, the liquidformulation is spray-dried to produce a dry powder.

In yet a further aspect, the invention provides a method for increasingthe aerosol performance of an active-agent containing formulationsuitable for administration to the lung. According to the method, a di-or tripeptide comprising at least two leucines is incorporated into aformulation comprising an active agent. The resulting compositioncomprising the active agent and the di- or tripeptide possesses anemitted dose that is increased over the emitted dose of a compositionhaving the same components but absent the di- or tripeptide. In oneembodiment, the method results in a liquid composition suitable foraerosolized administration to the lung; in an alternative embodiment,the method results in a dry powdered composition suitable foraerosolized administration to the lung.

Yet another aspect of the invention is directed to a method for deliveryof a dry powder composition to the lungs of a mammalian subject byadministering by inhalation the compositions of the invention aspreviously described, in aerosolized form.

These and other objects and features of the invention will become morefully apparent when the following detailed description is read inconjunction with the accompanying figures and examples.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The following terms as used herein have the meanings indicated.

“Active agent” as described herein includes any agent, drug, compound,composition of matter or mixture which provides some pharmacologic,often beneficial, effect that can be demonstrated in-vivo or in vitro.This includes foods, food supplements, nutrients, nutriceuticals, drugs,vaccines, antibodies, vitamins, and other beneficial agents. As usedherein, these terms further include any physiologically orpharmacologically active substance that produces a localized or systemiceffect in a patient.

“Amino acid” refers to any compound containing both an amino group and acarboxylic acid group. Although the amino group most commonly occurs atthe position adjacent to the carboxy function, the amino group may bepositioned at any location within the molecule. The amino acid may alsocontain additional functional groups, such as amino, thio, carboxyl,carboxamide, imidazole, etc. An amino acid may be synthetic or naturallyoccurring, and may be used in either its racemic or optically active(D-, or L-) form.

“Leucine”, whether present as a single amino acid or as an amino acidcomponent of a peptide, refers to the amino acid leucine, which may be aracemic mixture or in either its D- or L-form, as well as modified formsof leucine (i.e., where one or more atoms of leucine have beensubstituted with another atom or functional group) in which thedispersibility-enhancing effect of the modified amino acid or peptide issubstantially unchanged or unimproved over that of the unmodifiedmaterial.

“Dipeptide”, also referred to herein as a dimer, refers to a peptidecomposed of two amino acids.

“Tripeptide”, also referred to herein as a trimer, refers to a peptidecomposed of three amino acids.

A “surface active” material is one having surface activity (measured,e.g., by surface tensiometry), as characterized by its ability to reducethe surface tension of the liquid in which it is dissolved. Surfacetension, which is associated with the interface between a liquid andanother phase, is that property of a liquid by virtue of which thesurface molecules exhibit an inward attraction.

Typically, in the context of the present invention, a surface activedipeptide or tripeptide is identified by preparing solutions of varyingconcentrations (from approximately 0.01% wt/vol (0.1 mg/ml) toapproximately 2% wt/vol (20 mg/ml) of the subject peptide in water, andmeasuring the surface tension of each of the solutions. A surface-activepeptide is one which, when present at any concentration in solution,though typically present in an amount greater than 0.25 mg/ml, iseffective to lower the surface tension of water from its control value.A peptide that is more surface active than another peptide is one whichdecreases the surface tension of water to a greater extent, when presentin the liquid at the same concentration and measured under the same setof experimental conditions.

“Dry powder” refers to a powder composition that typically contains lessthan about 20% moisture, preferably less than 10% moisture, morepreferably contains less than about 5-6% moisture, and most preferablycontains less than about 3% moisture, depending upon the particularformulation.

A dry powder that is “suitable for pulmonary delivery” refers to acomposition comprising solid (i.e., non-liquid) or partially solidparticles that are capable of being (i) readily dispersed in/by aninhalation device and (ii) inhaled by a subject so that a portion of theparticles reach the lungs to permit penetration into the alveoli. Such apowder is considered to be “respirable”.

“Aerosolized” or “aerosolizable” particles are particles which, whendispensed into a gas stream by either a passive or an active inhalationdevice, remain suspended in the gas for an amount of time sufficient forat least a portion of the particles to be inhaled by the patient, sothat a portion of the particles reaches the lungs.

“Emitted Dose” or “ED” provides an indication of the delivery of a drugformulation from a suitable inhaler device after a firing or dispersionevent. More specifically, for dry powder formulations, the ED is ameasure of the percentage of powder which is drawn out of a unit dosepackage and which exits the mouthpiece of an inhaler device. The ED isdefined as the ratio of the dose delivered by an inhaler device to thenominal dose (i.e., the mass of powder per unit dose placed into asuitable inhaler device prior to firing). The ED is anexperimentally-determined parameter, and is typically determined usingan in-vitro device set up which mimics patient dosing. To determine anED value, a nominal dose of dry powder, typically in unit dose form, isplaced into a suitable dry powder inhaler (such as that described inU.S. Pat. No. 5,785,049, assigned to Inhale Therapeutic Systems) whichis then actuated, dispersing the powder. The resulting aerosol cloud isthen drawn by vacuum from the device, where it is captured on a taredfilter attached to the device mouthpiece. The amount of powder thatreaches the filter constitutes the emitted dose. For example, for a 5 mgdry powder-containing dosage form placed into an inhalation device, ifdispersion of the powder results in the recovery of 4 mg of powder on atared filter as described above, then the emitted dose for the drypowder composition is: 4 mg (delivered dose)/5 mg (nominaldose)×100=80%. For non-homogenous powders, ED values provide anindication of the delivery of drug from an inhaler device after firingrather than of dry powder, and are based on amount of drug rather thanon total powder weight. Similarly for MDI and nebulizer dosage forms,the ED corresponds to the percentage of drug which is drawn from adosage form and which exits the mouthpiece of an inhaler device.

“Fine particle dose” or “FPD” is defined as the mass percent of powderparticles having an aerodynamic diameter less than 3.3 μm, typicallydetermined by measurement in an Andersen cascade impactor. Thisparameter provides an indication of the percent of particles having thegreatest potential to reach the deep lung of a patient for systemicuptake of a drug substance.

A “dispersible” or “dispersive” powder is one having an ED value of atleast about 30%, more preferably 40-50%, and even more preferably atleast about 50-60%.

“Mass median diameter” or “MMD” is a measure of mean particle size,since the powders of the invention are generally polydisperse (i.e.,consist of a range of particle sizes). MMD values as reported herein aredetermined by centrifugal sedimentation, although any number of commonlyemployed techniques can be used for measuring mean particle size (e.g.,electron microscopy, light scattering, laser diffraction).

“Mass median aerodynamic diameter” or “MMAD” is a measure of theaerodynamic size of a dispersed particle. The aerodynamic diameter isused to describe an aerosolized powder in terms of its settlingbehavior, and is the diameter of a unit density sphere having the samesettling velocity, in air, as the particle. The aerodynamic diameterencompasses particle shape, density and physical size of a particle. Asused herein, MMAD refers to the midpoint or median of the aerodynamicparticle size distribution of an aerosolized powder determined bycascade impaction, unless otherwise indicated.

“Pharmaceutically acceptable salt” includes, but is not limited to,salts prepared with inorganic acids, such as chloride, sulfate,phosphate, diphosphate, hydrobromide, and nitrate salts, or saltsprepared with an organic acid, such as malate, maleate, fumarate,tartrate, succinate, ethylsuccinate, citrate, acetate, lactate,methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate,salicylate and stearate, as well as estolate, gluceptate andlactobionate salts. Similarly, salts containing pharmaceuticallyacceptable cations include, but are not limited to, sodium, potassium,calcium, aluminum, lithium, and ammonium (including alkyl substitutedammonium).

“Pharmaceutically acceptable excipient or carrier” refers to anexcipient that may optionally be included in the compositions of theinvention, and taken into the lungs with no significant adversetoxicological effects to the subject, and particularly to the lungs ofthe subject.

“Pharmacologically effective amount” or “physiologically effectiveamount of a bioactive agent” is the amount of an active agent present inan aerosolizable composition as described herein that is needed toprovide a desired level of active agent in the bloodstream or at thesite of action (e.g., the lungs) of a subject to be treated to give ananticipated physiological response when such composition is administeredpulmonarily. The precise amount will depend upon numerous factors, e.g.,the active agent, the activity of the composition, the delivery deviceemployed, the physical characteristics of the composition, intendedpatient use (i.e., the number of doses administered per day), patientconsiderations, and the like, and can readily be determined by oneskilled in the art, based upon the information provided herein.

“Polymer” refers to a high molecular weight polymeric compound ormacromolecule built by the repitition of small, simple chemical units. Apolymer may be a biological polymer, i.e., is naturally occurring (e.g.,proteins, carbohydrates, nucleic acids) or a non-biological,synthetically-produced polymer (e.g., polyethylene glycols,polyvinylpyrrolidones, Ficolls, and the like), as well known in the art.

II. The Composition

The present invention is based upon the Applicants' discovery of a classof compounds, dipeptides and tripeptides containing two or moreleucines, which when incorporated into formulations for administrationto the lung, impart superior aerosol properties to the resultingformulations. Moreover, the Applicants have discovered, surprisinglythat, these di- and tripeptides are effective to significantly enhancethe dispersibility of the resulting formulations, irrespective of thetype of active agent present in the formulation. Thus, these di- andtripeptides can be employed in a wide variety of formulations, toincrease the aerosol performance of the resulting compositions, and insome cases, to provide aerosolizable formulations in situations where anaerosolizable formulation was previously unknown or unattainable. Thepresent invention, although directed in certain respects to dry powderformulations, is meant to encompass liquid formulations as well. Thecomponents of the formulations of the invention will now be described.

A. The Active Agent

An active agent for incorporation in the compositions described hereinmay be an inorganic or an organic compound, including, withoutlimitation, drugs which act on: the peripheral nerves, adrenergicreceptors, cholinergic receptors, the skeletal muscles, thecardiovascular system, smooth muscles, the blood circulatory system,synoptic sites, neuroeffector junctional sites, endocrine and hormonesystems, the immunological system, the reproductive system, the skeletalsystem, autacoid systems, the alimentary and excretory systems, thehistamine system, and the central nervous system. Suitable agents may beselected from, for example, hypnotics and sedatives, psychic energizers,tranquilizers, respiratory drugs, anticonvulsants, muscle relaxants,antiparkinson agents (dopamine antagnonists), analgesics,anti-inflammatories, antianxiety drugs (anxiolytics), appetitesuppressants, antimigraine agents, muscle contractants, anti-infectives(antibiotics, antivirals, antifungals, vaccines) antiarthritics,antimalarials, antiemetics, anepileptics, bronchodilators, cytokines,growth factors, anti-cancer agents, antithrombotic agents,antihypertensives, cardiovascular drugs, antiarrhythmics, antioxicants,anti-asthma agents, hormonal agents including contraceptives,sympathomimetics, diuretics, lipid regulating agents, antiandrogenicagents, antiparasitics, anticoagulants, neoplastics, antineoplastics,hypoglycemics, nutritional agents and supplements, growth supplements,antienteritis agents, vaccines, antibodies, diagnostic agents, andcontrasting agents. The active agent, when administered by inhalation,may act locally or systemically.

The active agent may fall into one of a number of structural classes,including but not limited to small molecules, peptides, polypeptides,proteins, polysaccharides, steroids, proteins capable of elicitingphysiological effects, nucleotides, oligonucleotides, polynucleotides,fats, electrolytes, and the like.

Examples of active agents suitable for use in this invention include butare not limited to calcitonin, erythropoietin (EPO), Factor VIII, FactorIX, ceredase, cerezyme, cyclosporin, granulocyte colony stimulatingfactor (GCSF), thrombopoietin (TPO), alpha-1 proteinase inhibitor,elcatonin, granulocyte macrophage colony stimulating factor (GMCSF),growth hormone, human growth hormone (HGH), growth hormone releasinghormone (GHRH), heparin, low molecular weight heparin (LMWH), interferonalpha, interferon beta, interferon gamma, interleukin-1 receptor,interleukin-2, interleukin-1 receptor antagonist, interleukin-3,interleukin-4, interleukin-6, luteinizing hormone releasing hormone(LHRH), factor IX insulin, pro-insulin, insulin analogues (e.g.,mono-acylated insulin as described in U.S. Pat. No. 5,922,675), amylin,C-peptide, somatostatin, somatostatin analogs including octreotide,vasopressin, follicle stimulating hormone (FSH), insulin-like growthfactor (IGF), insulintropin, macrophage colony stimulating factor(M-CSF), nerve growth factor (NGF), tissue growth factors, keratinocytegrowth factor (KGF), glial growth factor (GGF), tumor necrosis factor(TNF), endothelial growth factors, parathyroid hormone (PTH),glucagon-like peptide thymosin alpha 1, IIb/IIIa inhibitor, alpha-1antitrypsin, phosphodiesterase (PDE) compounds, VLA-4 inhibitors,bisphosponates, respiratory syncytial virus antibody, cystic fibrosistransmembrane regulator (CFTR) gene, deoxyreibonuclease (Dnase),bactericidal/permeability increasing protein (BPI), anti-CMV antibody,13-cis retinoic acid, macrolides such as erythromycin, oleandomycin,troleandomycin, roxithromycin, clarithromycin, davercin, azithromycin,flurithromycin, dirithromycin, josamycin, spiromycin, midecamycin,leucomycin, miocamycin, rokitamycin, andazithromycin, and swinolide A;fluoroquinolones such as ciprofloxacin, ofloxacin, levofloxacin,trovafloxacin, alatrofloxacin, moxifloxicin, norfloxacin, enoxacin,grepafloxacin, gatifloxacin, lomefloxacin, sparfloxacin, temafloxacin,pefloxacin, amifloxacin, fleroxacin, tosufloxacin, prulifloxacin,irloxacin, pazufloxacin, clinafloxacin, and sitafloxacin,aminoglycosides such as gentamicin, netilmicin, paramecin, tobramycin,amikacin, kanamycin, neomycin, and streptomycin, vancomycin,teicoplanin, rampolanin, mideplanin, colistin, daptomycin, gramicidin,colistimethate, polymixins such as polymixin B, capreomycin, bacitracin,penems; penicillins including penicllinase-sensitive agents likepenicillin G, penicillin V, penicllinase-resistant agents likemethicillin, oxacillin, cloxacillin, dicloxacillin, floxacillin,nafcillin; gram negative microorganism active agents like ampicillin,amoxicillin, and hetacillin, cillin, and galampicillin; antipseudomonalpenicillins like carbenicillin, ticarcillin, azlocillin, mezlocillin,and piperacillin; cephalosporins like cefpodoxime, cefprozil, ceftbuten,ceftizoxime, ceftriaxone, cephalothin, cephapirin, cephalexin,cephradrine, cefoxitin, cefamandole, cefazolin, cephaloridine, cefaclor,cefadroxil, cephaloglycin, cefuroxime, ceforanide, cefotaxime,cefatrizine, cephacetrile, cefepime, cefixime, cefonicid, cefoperazone,cefotetan, cefmetazole, ceftazidime, loracarbef, and moxalactam,monobactams like aztreonam; and carbapenems such as imipenem, meropenem,pentamidine isethiouate, albuterol sulfate, lidocaine, metaproterenolsulfate, beclomethasone diprepionate, triamcinolone acetamide,budesonide acetonide, fluticasone, ipratropium bromide, flunisolide,cromolyn sodium, ergotamine tartrate and where applicable, analogues,agonists, antagonists, inhibitors, and pharmaceutically acceptable saltforms of the above. In reference to peptides and proteins, the inventionis intended to encompass synthetic, native, glycosylated,unglycosylated, pegylated forms, and biologically active fragments andanalogs thereof.

Active agents for use in the invention further include nucleic acids, asbare nucleic acid molecules, vectors, associated viral particles,plasmid DNA or RNA or other nucleic acid constructions of a typesuitable for transfection or transformation of cells, i.e., suitable forgene therapy including antisense. Further, an active agent may compriselive attenuated or killed viruses suitable for use as vaccines. Otheruseful drugs include those listed within the Physician's Desk Reference(most recent edition).

The amount of active agent in the formulation will be that amountnecessary to deliver a therapeutically effective amount of the activeagent per unit dose to achieve the desired result. In practice, thiswill vary widely depending upon the particular agent, its activity, theseverity of the condition to be treated, the patient population, dosingrequirements, and the desired therapeutic effect. The composition willgenerally contain anywhere from about 1% by weight to about 99% byweight active agent, typically from about 2% to about 95% by weightactive agent, and more typically from about 5% to 85% by weight activeagent, and will also depend upon the relative amounts of additivescontained in the composition. The compositions of the invention areparticularly useful for active agents that are delivered in doses offrom 0.001 mg/day to 100 mg/day, preferably in doses from 0.01 mg/day to75 mg/day, and more preferably in doses from 0.10 mg/day to 50 mg/day.

It is to be understood that more than one active agent may beincorporated into the formulations described herein and that the use ofthe term “agent” in no way excludes the use of two or more such agents.

B. Dispersibility-Enhancing Peptides

Compositions of the invention will include one or more di- ortripeptides containing two or more leucine residues. As discussed above,the invention is based upon the Applicants' discovery thatdi-leucyl-containing dipeptides (e.g., dileucine) and tripeptides aresuperior in their ability to increase the dispersibility of powderedcompositions, and, as demonstrated in the Examples, are unexpectedlybetter than leucine in improving aerosol performance.

Di-leucyl containing tripeptides for use in the invention aretripeptides having the formula, X-Y-Z, where at least X and Y or X and Zare leucyl residues (i.e., the leucyl residues can be adjacent to eachother (at the 1 and 2 positions), or can form the ends of the trimer(occupying positions 1 and 3). The remaining amino acid contained in thetrimer can be any amino acid as defined in section I above. Suitable areamino acids such as glycine (gly), alanine (ala), valine (val), leucine(leu), isoleucine (ile), methionine (met), proline (pro), phenylalanine(phe), trytophan (trp), serine (ser), threonine (thr), cysteine (cys),tyrosine (tyr), asparagine (asp), glutamic acid (glu), lysine (lys),arginine (arg), histidine (his), norleucine (nor), and modified farmsthereof. Preferably, for di-leucyl containing trimers, the third aminoacid component of the trimer is one of the following: leucine (leu),valine (val), isoleucine (ile), tryptophan (trp) alanine (ala),methionine (met), phenylalanine (phe), tyrosine (tyr), histidine (his),and proline (pro). Exemplary trimers for use in the invention includebut are not limited to the following: leu-leu-gly, leu-leu-ala,leu-leu-val, leu-leu-leu, leu-leu-ile, leu-leu-met, leu-leu-pro,leu-leu-phe, leu-leu-trp, leu-leu-ser, leu-leu-thr, leu-leu-cys,leu-leu-tyr, leu-leu-asp, leu-leu-glu, leu-leu-lys, leu-leu-arg,leu-leu-his, leu-leu-nor, leu-gly-leu, leu-ala-leu, leu-val-leu,leu-ile-leu, leu-met-leu, leu-pro-leu, leu-phe-leu, leu-trp-leu,leu-ser-leu, leu-thr-leu, leu-cys-leu, leu-tyr-leu, leu-asp-leu,leu-glu-leu, leu-lys-leu, leu-arg-leu, leu-his-leu, and leu-nor-leu.Particularly preferred peprides are dileucine and trileucine.

Although less preferred due to their limited solubility in water,additional dispersibility enhancing peptides for use in the inventionare 4-mers and 5-mers containing two or more leucine residues. Theleucine residues may occupy any position within the peptide, and theremaining (i.e., non-leucyl) amino acids positions are occupied by anyamino acid as described above, provided that the resulting 4-mer or5-mer has a solubility in water of at least about 1 mg/ml. Preferably,the non-leucyl amino acids in a 4-mer or 5-mer are hydrophilic aminoacids such as lysine, to thereby increase the solubility of the peptidein water.

Also preferred are di- and tripeptides having a glass transitiontemperature greater than about 40° C.

Preferred di- and tripeptides for use in the present invention are thosepeptides that are surface active. As can be seen from the surfacetension data in Example 1, dileucine and trileucine are extremelyeffective, even when present in low concentrations, at significantlydepressing the surface tension of water. Moreover, in examining thesurface tension results results in Table 5 (extrapolated values), it canbe seen that dipeptides and tripeptides containing two or more leucineshave a much greater surface activity than dipeptides and tripeptidescomposed of fewer than two leucyl residues. Due to their highly surfaceactive nature, the di- and tripeptides of the invention, when containedin dry powder compositions, tend to concentrate on the surface of thepowder particles, thereby imparting to the resulting particles highdispersivities. This feature of the powders, i.e., a surface enrichedwith the di- or tripeptide, is illustrated by the ESCA data provided inExample 9.

Surprisingly, the addition of the representative tripeptide, trileucine,to a calcitonin formulation was effective to nearly double the ED valueof the resulting powder (Example 4). This result is surprising becausecalcitonin itself is a surface active protein. Thus, the incorporationof another surface active material such as trileucine was not expectedto significantly improve the dispersivity of the composition. Results incontrast to this expectation indicated that surface activity alone isnot sufficient to significantly increase dispersibility, and furtherdemonstrated the unusual and beneficial properties of theleucyl-containing peptides of the invention, particularly in enhancingaerosol performance.

Generally, the compositions of the invention will contain from about 1%to about 99% by weight di- or tripeptide, preferably from about 2% toabout 75% by weight di- or tripeptide, and even more preferably fromabout 5% to about 50% by weight di- or tripeptide. Typically, theoptimal amount of di- or tripeptide is determined experimentally, i.e.,by preparing compositions containing varying amounts of di- ortripeptide (ranging from low to high), examining the dispersibilities ofthe resulting compositions as described herein, and further exploringthe range at which optimal aerosol performance is attained. Such methodswere employed in several of the Examples (Example 3, Example 4, Example5, Example 6). Generally, for trileucine containing dry powderformulations, an optimal amount of trileucine appears to be around22-25% by weight.

C. Additional Carriers and Excipients

In addition to the active agent and di- or tripeptide, compositions ofthe invention may optionally include one or more pharmaceuticalexcipients which are suitable for pulmonary administration. Theseexcipients, if present, are generally present in the composition inamounts ranging from about 0.01% to about 95% percent by weight,preferably from about 0.5 to about 80%, and more preferably from about 1to about 60% by weight. Preferably, such excipients will, in part, serveto further improve the features of the active agent composition, e.g.,by providing more efficient and reproducible delivery of the activeagent, improving the handling characteristics of powders (e.g.,flowability and consistency), and/or facilitating manufacturing andfilling of unit dosage forms. In particular, excipient materials canoften function to further improve the physical and chemical stability ofthe active agent, minimize the residual moisture content and hindermoisture uptake, and to enhance particle size, degree of aggregation,particle surface properties (i.e., rugosity), ease of inhalation, andthe targeting of particles to the lung. The excipient(s) may also servesimply as bulking agents when it is desired to reduce the concentrationof active agent in the formulation.

Pharmaceutical excipients and additives useful in the presentcomposition include but are not limited to amino acids, peptides,proteins, non-biological polymers, biological polymers, carbohydrates(e.g., sugars, derivatized sugars such as alditols, aldonic acids,esterified sugars, and sugar polymers), which may be present singly orin combination. Suitable excipients are those provided in InhaleTherapeutic Systems' International Publication No. WO 96/32096. Alsopreferred are excipients having glass transition temperatures (Tg),above about 35° C., preferably above about 40° C., more preferably above45° C., most preferably above about 55° C.

Exemplary protein excipients include albumins such as human serumalbumin (HSA), recombinant human albumin (rHA), gelatin, casein,hemoglobin, and the like. Suitable amino acids (outside of thedileucyl-peptides of the invention), which may also function in abuffering capacity, include alanine, glycine, arginine, betaine,histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine,isoleucine, valine, methionine, phenylalanine, aspartame, tyrosine,tryptophan, and the like. Preferred are amino acids and polypeptidesthat function as dispersing agents. Amino acids falling into thiscategory include hydrophobic amino acids such as leucine, valine,isoleucine, tryptophan, alanine, methionine, phenylalanine, tyrosine,histidine, and proline. Dispersibility-enhancing peptide excipientsinclude dimers, trimers, tetramers, and pentamers comprising one or morehydrophobic amino acid components such as those described above.

Carbohydrate excipients suitable for use in the invention include, forexample, monosaccharides such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol), pyranosyl sorbitol, myoinositol and the like.

The compositions may also include a buffer or a pH adjusting agent,typically a salt prepared from an organic acid or base. Representativebuffers include organic acid salts of citric acid, ascorbic acid,gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid,or phthalic acid, Tris, tromethamine hydrochloride, or phosphatebuffers.

The compositions of the invention may also include polymericexcipients/additives, e.g., polyvinylpyrrolidones, derivatizedcelluloses such as hydroxymethylcellulose, hydroxyethylcellulose, andhydroxypropylmethylcellulose, Ficolls (a polymeric sugar),hydroxyethylstarch, dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin and sulfobutylether-β-cyclodextrin),polyethylene glycols, and pectin.

The compositions may further include flavoring agents, taste-maskingagents, inorganic salts (e.g., sodium chloride), antimicrobial agents(e.g., benzalkonium chloride), sweeteners, antioxidants, antistaticagents, surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN80”), sorbitan esters, lipids (e.g., phospholipids such as lecithin andother phosphatidylcholines, phosphatidylethanolamines), fatty acids andfatty esters, steroids (e.g., cholesterol), and chelating agents (e.g.,EDTA, zinc and other such suitable cations). Other pharmaceuticalexcipients and/or additives suitable for use in the compositionsaccording to the invention are listed in “Remington: The Science &Practice of Pharmacy”, 19^(th) ed., Williams & Williams, (1995), and inthe “Physician's Desk Reference”, 52^(nd) ed., Medical Economics,Montvale, N.J. (1998).

III. Formulation Types

The compositions described herein may be in powdered form or may beflowable liquids. Liquid formulations are preferably solutions in whichthe active drug is dissolved in a solvent (e.g., water, ethanol,ethanol-water, saline) and less preferably are colloidal suspensions.The liquid formulation may also be a solution or suspension of theactive agent in a low boiling point propellant.

Liquid formulations containing the disclosed dileucyl-containingpeptides are also highly dispersible, possessing high ED values.

IV. Preparing Dry Powders

Dry powder formulations are preferably prepared by spray drying. Spraydrying of the formulations is carried out, for example, as describedgenerally in the “Spray Drying Handbook”, 5^(th) ed., K. Masters, JohnWiley & Sons, Inc., NY, N.Y. (1991), and in Platz, R., et al.,International Patent Publication No. WO 97/41833 (1997), the contents ofwhich are incorporated herein by reference.

Active agents having a solubility in water of at least about 0.10 mg/ml(e.g., peptides, proteins, nucleotides and the like) can be sprayeddried from an aqueous solution. Utilizing this approach, the activeagent is first dissolved in water, optionally containing aphysiologically acceptable buffer. The pH range of activeagent-containing solutions is generally between about 4 and 11, withnearer neutral pHs being preferred, since such pHs may aid inmaintaining the physiological compatibility of the powder afterdissolution of powder within the lung. The aqueous formulation mayoptionally contain additional water-miscible solvents, such as acetone,alcohols and the like. Representative alcohols are lower alcohols suchas methanol, ethanol, propanol, isopropanol, and the like. The pre-spraydried solutions will generally contain solids dissolved at aconcentration from 0.01% (weight/volume) to about 20% (weight/volume),usually from 0.1% to 3% (weight/volume).

The solutions are then spray dried in a conventional spray drier, suchas those available from commercial suppliers such as Niro A/S (Denmark),Buchi (Switzerland) and the like, resulting in a dispersible, drypowder. Optimal conditions for spray drying the solutions will varydepending upon the formulation components, and are generally determinedexperimentally. The gas used to spray dry the material is typically air,although inert gases such as nitrogen or argon are also suitable.Moreover, the temperature of both the inlet and outlet of the gas usedto dry the sprayed material is such that it does not cause decompositionof the active agent in the sprayed material. Such temperatures aretypically determined experimentally, although generally, the inlettemperature will range from about 50° C. to about 200° C. while theoutlet temperature will range from about 30° C. to about 150° C.

Variations of the above are utilized for spray-drying formulations wherethe active agent is a hydrophobic drug. One such process is described inGordon, M. S., Lord, J. D., U.S. Pat. No. 5,985,248, assigned to InhaleTherapeutics Systems. In this method, a hydrophobic drug is dissolved inan organic solvent or co-solvent system, and the hydrophilic components(e.g., the leucyl-containing peptides and optional other excipients) areat least partially dissolved in the same organic solvent or co-solventsystem. The resulting solution is then spray-dried to form particles.Typically, the solubility of the active agent and the hydrophiliccomponent will govern the selection of the organic solvent system. Theorganic solvent is selected to provide a solubility for the hydrophiliccomponent of at least 1 mg/ml, and preferably at least 5 mg/ml, and asolubility for the hydrophobic drug of at least 0.01 mg/ml, preferablyat least 0.05 mg/ml.

Alternatively, the composition may be prepared by spray-drying asuspension, as described in Gordon, M. S., U.S. Pat. No. 5,976,574,assigned to Inhale Therapeutic Systems. In this method, the hydrophobicdrug is dissolved in an organic solvent, e.g., methanol, ethanol,isopropanol, acetone, heptane, hexane chloroform, ether, followed bysuspension of the hydrophilic excipient in the organic solvent to form asuspension. The suspension is then spray-dried to form particles.Preferred solvents, for both of the above spray-drying methods includealcohols, ethers, ketones, hydrocarbons, polar aprotic solvents, andmixtures thereof.

The dry powders of the invention may also be prepared by combiningaqueous solutions or suspensions of the formulation components andspray-drying them simultaneously in a spray-dryer, as described inGordon, M., U.S. Pat. No. 6,001,336, assigned to Inhale TherapeuticSystems. Alternatively, the dry powders may be prepared by preparing anaqueous solution of a hydrophilic excipient or additive, preparing anorganic solution of a hydrophobic drug, and spray drying the aqueoussolution and the organic solution simultaneously through a nozzle, e.g.,a coaxial nozzle, to form a dry powder, as described in Gordon, M., etal, International Publication Number WO 98/29096.

Alternatively, powders may be prepared by lyophilization, vacuum drying,spray freeze drying, super critical fluid processing, air drying, orother forms of evaporative drying. In some instances, it may bedesirable to provide the dry powder formulation in a form that possessesimproved handling/processing characteristics, e.g., reduced static,better flowability, low caking, and the like, by preparing compositionscomposed of fine particle aggregates, that is, aggregates oragglomerates of the above-described dry powder particles, where theaggregates are readily broken back down to the fine powder componentsfor pulmonary delivery, as described, e.g., in Johnson, K., et al., U.S.Pat. No. 5,654,007, 1997, incorporated herein by reference.

In another approach, dry powders may be prepared by agglomerating thepowder components, sieving the materials to obtain agglomerates,spheronizing to provide a more spherical agglomerate, and sizing toobtain a uniformly-sized product, as described, e.g., and in Ahlneck,C., et al., International PCT Publication No. WO 95/09616, 1995,incorporated herein by reference.

Dry powders may also be prepared by blending, grinding, sieving or jetmilling formulation components in dry powder form.

Once formed, the dry powder compositions are preferably maintained underdry (i.e., relatively low humidity) conditions during manufacture,processing, and storage. Irrespective of the drying process employed,the process will preferably result in respirable, highly dispersibleparticles comprising an active agent and a dileucyl-containing dimer ortrimer.

V. Features of Dry Powder Formulations

Powders of the invention are further characterized by several features,most notably, (i) consistently high dispersivities, which aremaintained, even upon storage (Example 8), (ii) small aerodynamicparticles sizes (MMADs), (iii) improved fine particle dose values, i.e.,powders having a higher percentage of particles sized less than 3.3microns MMAD, all of which contribute to the improved ability of thepowder to penetrate to the tissues of the lower respiratory tract (i.e.,the alveoli) for either localized or systemic treatment. These physicalcharacteristics of the di-leucyl peptide-containing dry powders, to bedescribed more fully below, are important in maximizing the efficiencyof aerosolized delivery of such powders to the deep lung.

Dry powders of the invention are composed of aerosolizable particleseffective to penetrate into the lungs. The particles of the inventionhave a mass median diameter (MMD) of less than about 20 μm, preferablyless than about 10 μm, more preferably less than about 7.5 μm, and mostpreferably less than about 4 μm, and usually are in the range of 0.1 μmto 5 μm in diameter. Preferred powders are composed of particles havingan MMD from about 0.2 to 4.0 μm. In some cases, the powder will alsocontain non-respirable carrier particles such as lactose, where thenon-respirable particles are typically greater than about 40 microns insize.

The powders of the invention are further characterized by an aerosolparticle size distribution less than about 10 μm mass median aerodynamicdiameter (MMAD), and preferably less than 4.0 μm. The mass medianaerodynamic diameters of the powders will characteristically range fromabout 0.1-10 μm, preferably from about 0.2-5.0 μm MMAD, more preferablyfrom about 1.0-4.0 μm MMAD, and even more preferably from about 1.5 to3.5 μm. Illustrative MMAD values for exemplarydi-leucyl-peptide-containing powder compositions are provided inExamples 2, 3, 4, 5, and 6. Several of these examples demonstrate animprovement in aerosol particle size distribution achieved uponincorporation of a di-leucyl di- or tripeptide into the formulation.

The powders of the invention may further be characterized by theirdensities. The powder will generally possess a bulk density from about0.1 to 10 g/cubic centimeter, preferably from about 0.1-2 g/cubiccentimeter, and more preferably from about 0.15-1.5 g/cubic centimeter.

The powders will generally have a moisture content below about 20% byweight, usually below about 10% by weight, and preferably below about 6%by weight. Such low moisture-containing solids tend to exhibit a greaterstability upon packaging and storage.

One of the most striking features of the compositions of the inventionis their dispersibility, as indicated by the ED value. The presence ofthe di-leucyl peptide in the formulations is effective to provideformulations having significantly improved dispersibilities. Generally,the emitted dose (ED) of these powders is greater than 30%, and usuallygreater than 40%. More preferably, the ED of the powders of theinvention is greater than 50%, and is often greater than 55%. In fact,in looking at the Examples, di-leucyl-peptide containing powderstypically possess optimized ED values as high as 80% or above. Moreover,the Examples further illustrate that the incorporation of a di-leucyldi- or tripeptide into a variety of active agent formulations waseffective, in all cases, to increase the ED value of the resultantcompositions, and in some instances, as much as doubling its value.Moreover, this effect was observed for both protein and small moleculeactive agent powders.

An additional measure for characterizing the overall aerosol performanceof a dry powder is the fine particle dose (FPD), which describes thepercentage of powder having an aerodynamic diameter less than 3.3microns. The powders of the invention are particularly well suited forpulmonary delivery, and possess FPF values ranging from about 35%-85%.Such powders contain at least about 35 percent of aerosol particle sizesbelow 3.3 μm to about 0.5 μm and are thus extremely effective whendelivered in aerosolized form, in reaching the regions of the lung,including the alveoli.

The compositions described herein also possess good stability withrespect to both chemical stability and physical stability, i.e., aerosolperformance, over time (Example 8). Generally, with respect to chemicalstability, the active agent contained in the formulation will degrade byno more than about 10% over a time course of three months, preferably byno more than about 7%, and more preferably by no more than 5%, uponstorage of the composition under ambient conditions. As illustrated bythe exemplary PTH formulation in Example 8, storage under acceleratedstability conditions (40° C., ambient humidity) for over a period of 3months (12 weeks) resulted in the degradation of only 2.3% protein (froman initial value of 97.1% purity to 94.8% purity). Since acceleratedtemperatures result in an increase in reaction rate, one can concludethat storage of the same composition under ambient conditions wouldresult in a degradation rate less than 2.3%, further pointing to thechemical stability of the present compositions.

With respect to aerosol performance, compositions of the invention aregenerally characterized by a drop in emitted dose of no more than about20%, preferably no more than about 15%, and more preferably by no morethan about 10%, when stored under ambient conditions for a period ofthree months. In looking at the results in Example 8, an exemplaryPTH-trileucine formulation exhibited essentially no change, and inparticular, no diminishment, in aerosol properties (MMAD, FPD, ED) uponstorage under accelerated stability conditions (40° C., ambienthumidity).

Another preferred feature of particulate compositions of the inventionis an enrichment of the di-leucyl di- or tripeptide on the surface ofthe particles, as indicated by the results in Example 9.

The improvement in aerosol properties discovered for di-leucyl di- andtripeptide-containing composition (i.e., greatly enhanceddispersibilities, reduced fine particle dose values, smaller aerodynamicdiameters), can result in several related advantages, such as: (i)reducing costly drug loses to the inhalation device, since more powderis aerosolized and is therefore available for inhalation by a subject;(ii) reducing the amount of dry powder required per unit dose, due tothe high efficiency of aerosolization of powder, (iii) reducing thenumber of inhalations per day by increasing the amount of aerosolizeddrug reaching the lungs of a subject.

VI. Administration of the Composition

The formulations described herein may be delivered using any suitabledry powder inhaler (DPI), i.e., an inhaler device that utilizes thepatient's inhaled breath as a vehicle to transport the dry powder drugto the lungs. Preferred are Inhale Therapeutic Systems' dry powderinhalation devices as described in Patton, J. S., et al., U.S. Pat. No.5,458,135 (1995) Smith, A. E., et al., U.S. Pat. No. 5,740,794 (1998);and in Smith, A. E., et. al., U.S. Pat. No. 5,785,049 (1998), hereinincorporated by reference.

When administered using a device of this type, the powder is containedin a receptacle having a puncturable lid or other access surface,preferably a blister package or cartridge, where the receptable maycontain a single dosage unit or multiple dosage units. Convenientmethods for filling large numbers of cavities (i.e., unit dose packages)with metered doses of dry powder medicament are described, e.g., inParks, D. J., et al., WO 97/41031 (1997) incorporated herein byreference.

Also suitable for delivering the powders described herein are dry powderinhalers of the type described, for example, in Cocozza, S., et al.,U.S. Pat. No. 3,906,950 (1974), and in Cocozza, S., et al., U.S. Pat.No. 4,013,075 (1997), incorporated herein by reference, wherein apremeasured dose of dry powder for delivery to a subject is containedwithin a hard gelatin capsule.

Other dry powder dispersion devices for pulmonarily administering drypowders include those described, for example, in Newell, R. E., et al,European Patent No. EP 129985 (1988); in Hodson, P. D., et al, EuropeanPatent No. EP 472598 (1996); in Cocozza, S., et al., European Patent No.EP 467172 (1994), and in Lloyd, L. J. et al., U.S. Pat. No. 5,522,385(1996), incorporated herein by reference. Also suitable for deliveringthe dry powders of the invention are inhalation devices such as theAstra-Draco “TURBUHALER”. This type of device is described in detail inVirtanen, R., U.S. Pat. No. 4,668,281 (1987); in Wetterlin, K., et alU.S. Pat. No. 4,667,668 (1987); and in Wetterlin, K., et al. U.S. Pat.No. 4,805,811 (1989), all of which are incorporated herein by reference.Other suitable devices include dry powder inhalers such as theRotahaler® (Glaxo), Discus® (Glaxo), Spiros™ inhaler (DuraPharmaceuticals), and the Spinhaler® (Fisons). Also suitable are deviceswhich employ the use of a piston to provide air for either entrainingpowdered medicament, lifting medicament from a carrier screen by passingair through the screen, or mixing air with powder medicament in a mixingchamber with subsequent introduction of the powder to the patientthrough the mouthpiece of the device, such as described in Mulhauser,P., et al, U.S. Pat. No. 5,388,572 (1997), incorporated herein byreference.

Dry powders may also be delivered using a pressurized, metered doseinhaler (MDI), e.g., the Ventolin® metered dose inhaler, containing asolution or suspension of drug in a pharmaceutically inert liquidpropellant, e.g., a chlorofluorocarbon or fluorocarbon, as described inLaube, et al., U.S. Pat. No. 5,320,094 (1994), and in Rubsamen, R. M.,et al, U.S. Pat. No. 5,672,581 (1994), both incorporated herein byreference. Alternatively, the powders described herein may be dissolvedor suspended in a solvent, e.g., water, ethanol, or saline, andadministered by nebulization. Nebulizers for delivering an aerosolizedsolution include the AERx™ (Aradigm), the Ultravent® (Mallinkrodt), andthe Acorn II® (Marquest Medical Products).

Prior to use, dry powders are generally stored under ambient conditions,and preferably are stored at temperatures at or below about 25° C., andrelative humidities (RH) ranging from about 30 to 60%. More preferredrelative humidity conditions, e.g., less than about 30%, may be achievedby the incorporation of a dessicating agent in the secondary packagingof the dosage form.

VII. Utility

The compositions of the invention are useful, when administeredpulmonarily in a therapeutically effective amount to a mammaliansubject, for treating or preventing any condition responsive to theadministration of an active agent as described in section II.A above.

The following examples are illustrative of the present invention, andare not to be construed as limiting the scope of the invention.Variations and equivalents of this example will be apparent to those ofskill in the art in light of the present disclosure, the drawings andthe claims herein.

All articles, books, patents and other publications referenced hereinare hereby incorporated by reference in their entirety.

EXAMPLES Materials and Methods

A. Materials.

Ciprofloxacin Hydrochloride (Neuland Laboratories, India).

Gentamicin Sulfate (H&A (Canada) Industrial)

Netilmicin Sulfate (Scientific Instruments And Technology)

L-Leucine (Aldrich, St. Louis, Mo.)

Hydrochloric Acid (J. T. Baker, Phillipsburg, N.J.)

Sodium Hydroxide 0.1N Volumetric Solution (J. T. Baker, Phillipsburg,N.J.)

Ethanol, 200 proof (USP/NF, Spectrum Chemical Mfg. Corp., New Brunswick,N.J.)

Methanol (HPLC grade, EM Industries, Gibbstown, N.J.)

S. calcitonin (Bachem California Inc, USA Torrance, Calif.).

Trileucine (Bachem California Inc, USA Torrance, Calif.).

Other amino acids used in surface tension experiments were obtained fromSigma St. Louis, Mo.

B. Methods.

Particle Size Measurements (Horiba)

Mass median diameters (MMD) of the powders were measured using a HoribaCAPA-700 particle size analyzer (Horiba Instruments inc., Irvine,Calif.). Measurements were based upon centrifugal sedimentation ofdispersed particles in suspending medium. Mass median diameter, which isbased on the particle's Stokes' diameter, was calculated using theparticle density and the density and viscosity of the suspending medium.

The density of the powder was set as 1.5 g/cm³ for all powders. (Thisnominal value was used for all powders analyzed and is within a rangethat is typical for spray dried powders). Particle size measurementswere conducted with about 5-10 mg powder suspended in 5 ml SedisperseA-11 (Micromeritics, Norcross, Ga.) and dispersed by sonication for 10minutes. The range over which particle size data was gathered was set to0.4 to 10.0 μm.

Aerodynamic Particle Size Measurements

Andersen Cascade Impactor. An Andersen cascade impactor (a sieve-likeapparatus with a series of stages that capture particles on plates byinertial impaction according to their size) was used to determine theMMAD and particle size distribution of aerosolized powder formulationsin an air stream. The plates were weighed before and after testing andthe mass of powder deposited on the plate of each stage was determined.Unless otherwise indicated, studies were undertaken using a traditionalAndersen cascade impactor having eight stages (from top to bottom stages0 to 7) with cut-off sizes ranging from 9.0 to 0.4 μm, and a finalfilter stage that traps particles <0.4 μm when operated at a flow rateof 28.3 L/min. The device test set-up was similar to the ED test, exceptthat the cascade impactor and a USP (United States Pharmacopia) throat(USP 23, chapter <601>) were attached to the device mouthpiece ratherthan to a filter. Multiple dispersions were typically conducted for eachcascade impaction run to achieve gravimetrically accurate data.

Andersen Short Stack (SS) Method. In the SS method, the order in whichthe stages were placed were altered from the conventional Andersencascade impactor set-up as described above. From the top, stage 0 wasutilized for inlet cone attachment to connect the throat. Stage 3 waspositioned next, beneath stage 0, followed by the filter stage (stageF). The powder-containing airstream passes only through stages 0 and 3;air (but not powder) flows through the other stages, which are placedunder stage F to hold the remainder of the assembly in place. Apre-weighed filter was placed on stage F and captured particles <3.3 μm.A second filter was placed on an inverted plate under stage 3, andcaptured particles >3.3 μm. For the studies described herein, one BP(blister pack) containing 2 mg of powder composition was dispersed in anaerosol delivery device and a vacuum was pulled at 28.3 L/min as per USPmethodology. This process was then repeated two times for a target massof 6 mg per run. The filters were then removed and weighed to determinethe amount of powder deposited.

Example 1 Surface Activity of Di- and Tripeptides

The surface tension of several representative dipeptides, tripeptides,and proteins was measured at 25° C. and 45° C. to provide an indicationof their relative surface activities. Surface tension measurements werecarried out using a Kruss Processor Tensiometer-K12 with theWilhelmy-method (Plate method).

Solutions were prepared by dissolving either 0.05%, 0.2%, or 0.6%peptide/protein (by weight) along with an appropriate amount ofraffinose by weight to provide final solutions having a 1.0% by weightsolids content. Surface tension measurements at 25° C. and 45° C. werethen obtained for the test solutions at three different time points (49seconds, 100 seconds and 194 seconds). The results are shown in Tables1-5 below

Highly surface active peptides and proteins are those that are effectiveto lower the surface tension of water from its control value(s). As canbe seen in Tables 1-4, raffinose (which was added to each of thesolutions to bring the overall solids content to 1.0%) is non-surfaceactive, and thus does not impact the surface tension results obtainedfor each of the peptides/proteins.

In looking at the results below, it can be seen that highly surfaceactive peptides include the peptides, dileucine and trileucine. Thesepeptides were as effective as the highly surface active protein, salmoncalcitonin, at significantly lowering the surface tension of water.Trileucine was effective at lowering the surface tension of water to agreater extent at higher concentrations (see, for example, data for0.05%, 0.2% and 0.6% by weight tri-leucine). In comparison to trileucineand dileucine, the dimer of isoleucine and the dimer and trimer ofvaline were not particularly effective at lowering the surface tensionof water.

This method can be used to identify additional surface active di- andtri-peptides suitable for use in the dry powders of the invention.

TABLE 1 Surface Tension Measurements SAMPLE ST, mN/m time,s ST, mN/mtime, s St, mN/m time, s water blank-1 72.6 49 72.6 100 72.6 194 waterblank-2 72.5 49 72.5 100 72.4 194 water blank-3 72.5 49 72.4 100 72.4194 1% raffinose-1 72 49 72 100 72 194 1% raffinose-2 72 49 72 100 72194 1% raffinose-3 72 49 72 100 72 194 0.2% tri-alanine-1 72.4 49 72.4100 72.3 194 0.2% tri-alanine-2 72.2 49 72.2 100 72.2 194 0.2%tri-alanine-3 72.3 49 72.2 100 72.2 194 0.2% tri-glutamate-1 72.1 4972.1 100 72.1 194 0.2% tri-glutamate-2 72.4 49 72.4 100 72.4 194 0.2%tri-glutamate-3 72.4 49 72.4 100 72.3 194 0.2% di-alanine-1 72 49 72 10071.9 194 0.2% di-alanine-2 72 49 71.9 100 71.9 194 0.2% di-alanine-372.1 49 72.1 100 72.1 194 0.2% di-leucine-1 58.4 49 58.1 100 57.9 1940.2% di-leucine-2 58.7 49 58.3 100 58.2 194 0.2% di-leucine-3 60.1 4959.8 100 59.7 194 0.2% tri-leucine-1 51 49 50.9 100 50.9 194 0.2%tri-leucine-2 51 49 50.8 100 50.7 194 0.2% tri-leucine-3 51 49 50.8 10050.7 194 0.2% sal. Calcitonin-1 48.7 49 48.6 100 48.5 194 0.2%sal.calcitonin-2 48.4 49 48.4 100 48.4 194 0.2% sal.calcitonin-3 48.4 4948.4 100 48.4 194 Measurements conducted at 25° C. The 0.2% (wt/vol)solutions additionally contain raffinose to form solutions having atotal solids content of 1% (wt/vol).

TABLE 2 Surface Tension Measurements SAMPLE ST, mN/m time, s ST, mN/mtime, s St, mN/m time, s water blank-1 72 49 71.8 100 71.7 194 waterblank-2 72.2 49 72.2 100 72.2 194 water blank-3 71.5 49 71.6 100 71.6194 0.2% di-isoleucine-1 67.6 49 67.2 100 67 194 0.2% di-isoleucine-2 6849 67.8 100 67.6 194 0.2% di-isoleucine-3 67.7 49 71.6 100 71.6 194 0.2%di-valine-1 71.7 49 71.6 100 71.6 194 0.2% di-valine-2 71.6 49 71.6 10071.6 194 0.2% di-valine-3 71.7 49 71.6 100 71.6 194 0.2% tri-valine-168.8 49 68.8 100 68.8 194 0.2% tri-valine-2 68.8 49 68.7 100 68.7 1940.2% tri-valine-3 68.7 49 68.7 100 68.7 194 Surface tension measurementsconducted at 25° C. Solutions contained 0.20% (wt/vol) of one of:di-isoleucine, di-valine, or tri-valine and 0.80% (wt/vol) raffinose.

TABLE 3 Surface Tension Measurements SAMPLE ST, mN/m time, s ST, mN/mtime, s St, mN/m time, s 1% raffinose (pH4)-1 71.4 49 71.4 100 71.4 1941% raffinose (PH4)-2 71.1 49 71.1 100 71.1 194 1% raffinose (pH4)-3 71.149 71.1 100 71.1 194 1% raffinose(pH7)-1 71.1 49 71.1 100 71.1 194 1%raffinose (pH7)-2 71.1 49 71.1 100 71.1 194 1% raffinose (pH7)-3 71.1 4971.1 100 71.1 194 water blank-1 72.1 49 72 100 72 194 water blank-2 72.249 72.1 100 72 194 water blank-3 72.2 49 72.1 100 72 194 0.05%leu3(pH4)-1 59.9 49 59.8 100 59.7 194 0.05% leu3(pH4)-2 60.4 49 60.3 10060.2 194 0.05% leu3(pH4)-3 60.4 49 60.3 100 60.2 194 0.2% leu3(pH4)-151.4 49 51.2 100 51.1 194 0.2% leu3(pH4)-2 51.4 49 51.3 100 51.2 1940.2% leu3(pH4)-3 51.4 49 51.2 100 51.1 194 0.6% leu3(pH4)-1 44.2 49 44.1100 44 194 0.6% leu3(pH4)-2 44.3 49 44.2 100 44.2 194 0.6% leu3(pH4)-344.2 49 44.2 100 44.1 194 0.05% leu3(pH7)-1 60.1 49 59.8 100 59.7 1940.05% leu3(pH7)-2 60 49 59.8 100 59.7 194 0.05% leu3(pH7)-3 60.2 49 60100 59.8 194 0.2% leu3(pH7)-1 51 49 50.8 100 50.7 194 0.2% leu3(pH7)-250.9 49 50.7 100 50.6 194 0.2% leu3(pH7)-3 50.7 49 50.5 100 50.4 1940.6% leu3(pH7)-1 43.7 49 43.7 100 43.6 194 0.6% leu3(pH7)-2 43.8 49 43.7100 43.7 194 0.6% leu3(pH7)-3 43.8 49 43.7 100 43.7 194 water blank-571.7 49 71.7 100 71.6 194 water blank-6 72.2 49 72.1 100 72.1 194Surface tension measurements measured at 25° C. The trileucineformulations also contain raffinose to provide solutions having a totalsolids content of 1% (wt/vol).

TABLE 4 Surface Tension Measurements SAMPLE ST, mN/m time, s ST, mN/mtime, s St, mN/m time, s water blank-5 69.2 49 69.2 100 69.2 194 1%raffinose(pH4)-1 67.9 49 68 100 68 194 1% raffinose (pH4)-2 68.2 49 68.2100 68.2 194 1% raffinose (pH4)-3 68 49 68 100 68.1 194 1% raffinose(pH7)-1 68.3 49 68.3 100 68.3 194 1% raffinose (pH7)-2 68.4 49 68.4 10068.4 194 1% raffinose (pH7)-3 68.4 49 68.4 100 68.4 194 Leu3formulations contain raffinose to make 1% total solids content 0.05%leu3(pH4)-1 57.1 49 57 100 57 194 0.05% leu3(pH4)-2 58.1 49 57.9 10057.8 194 0.05% leu3(pH4)-3 58 49 57.8 100 57.8 194 0.2% leu3(pH4)-1 47.949 47.5 100 47.4 194 0.2% leu3(pH4)-2 47.2 49 47.2 100 47.3 194 0.2%leu3(pH4)-3 47.9 49 47.3 100 47.1 194 0.6% leu3(pH4)-1 40.9 49 40.9 10040.8 194 0.6% leu3(pH4)-2 41.1 49 41 100 40.9 194 0.6% leu3(pH4)-3 41.149 41 100 40.8 194 0.05% leu3(Ph7)-1 58.5 49 58.4 100 58.4 194 0.05%leu3(pH7)-2 58.2 49 58.2 100 58.1 194 0.05% leu3(pH7)-3 58.2 49 58.1 10058.1 194 0.2% leu3(pH7)-1 58.5 49 58.4 100 58.4 194 0.2% leu3(pH7)-258.2 49 58.2 100 58.1 194 0.2% leu3(pH7)-3 58.2 49 58.1 100 58.1 194Surface tension measurements taken at 45° C. Tri-leucine-containingformulations also contain raffinose to provide a solution having a totalsolids content of 1%. Additional surface tension measurements wereobtained to determine dimers and trimers for use in the invention (i.e.,surface active dimers and trimers).

TABLE 5 Surface Tension of Representative Dimers and Trimers 25° C. 45°C. 25° C. 45° C. Extrapolated Extrapolated concentration Actual ActualValues Values SAMPLE mg/ml MEAN SD MEAN SD at 2 mg/ml at 2 mg/ml DimersLeu-2 13.60 46.6 0.6 42.7 0.3 60.4 52.4 4.53 54.9 0.5 48.2 0.1 1.51 61.50.6 53.2 0.1 Leu-Val 8.80 59.1 0.2 55.7 0.3 67.2 62.3 2.93 65 0.2 60.40.0 0.98 69.2 0.4 64.2 0.2 Leu-Tyr 6.40 62.2 0.1 59.5 0.3 68.3 67.3 2.1368.0 0.1 65.6 0.3 0.71 71.5 0 68.0 0.1 Val-Leu 7.80 68 0 63.5 0.2 69.865.3 2.60 69.5 0.1 65.0 0.2 0.87 70 0.5 65.5 0.0 Val-Ile 10.00 66.1 061.9 0.2 70.3 65.8 3.33 70.1 0.3 65.3 0.1 1.11 71.6 0.2 66.3 0.2 Leu TBD56.7 0.3 54.7 0.2 66.3 0.2 61.4 0.2 Trimers 70.8 0.2 64.2 0.0 Leu-Tyr-2.90 44.7 0.1 40.8 0.0 47.9 44.7 Leu 0.97 51.6 0.1 49.1 0.1 0.32 58.40.1 55.4 0.3 Leu-Phe- 6.10 41.5 0.2 39.3 0.0 48.3 46.2 Leu 2.03 48.3 046.2 0.1 0.68 54.7 0.1 53.6 0.1 Leu-3 6.10 42.4 0 38.9 0.2 49.7 46.32.03 49.7 0 46.3 0.3 0.68 56.9 0 52.8 0.6 Leu-Leu- 6.80 39.9 0.5 48.40.2 46.6 49.8 Ala 2.27 43.5 0.8 48.2 4.3 0.76 60.7 0.4 58.3 0.5 Ala-Val-8.70 55.7 0.2 53.8 0.0 65   58.9 Leu 2.90 62.8 0.5 57.7 0.2 0.97 67.50.5 60.3 0.1

As can be seen from the above, surface active dimers and trimers aremore effective when present at higher concentrations at lowering thesurface tension of water. As an example, at a concentration of 1.20mg/ml, the presence of trileucine was effective to lower the surfacetension of water from about 72 mN/m to 42 mN/s, while at a concentrationof 0.68 mg/ml, trileucine was effective at lowering the surface tensionof water to about 57 mN/m.

To normalize for concentration effects, surface tension values wereextrapolated to solutions having a concentration of 2 mg/ml (Table 5,columns 7 and 8). Looking first at the dimers, dileucine was moreeffective than any of the other dimers examined in reducing the surfacetension of water. Looking at data for the trimers, leu-tyr-leu is themost surface active of the trimers. Trimers containing, in addition totwo leucyl residues, a hydrophobic amino acid such as tyrosine,phenylalanine, leucine, or alanine, are more surface active than trimerscontaining fewer than two leucyl residues.

In summary, dimers and trimers containing two or more leucines wereeffective at significantly lowering the surface tension of water (e.g.,leu-try-ala, leu-phe-leu, leu-leu-leu, leu-leu-ala, and the like), andare preferred for use in the compositions of the invention.

Example 2 Aerosol Properties of a Parathyroid Hormone (PTH)-TrileucineDry Powder

Dry powders containing an illustrative active protein, parathyroidhormone, in combination with either leucine or tri-leucine, wereprepared. Also prepared was a dry powder absent either leucine ortrileucine, to demonstrate the notable improvement in aerosol propertiesupon addition of trileucine.

Representative PTH powders were prepared as follows.

A. Solution Formulation Preparation

Aqueous formulation solutions were prepared at a total solids content of1% (w/v). The pH of each solution was determined, and solutions werethen spray-dried. Table 6 lists the compositions of all pre-spray-driedPTH solutions.

B. Powder Processing: Spray Drying

Powders were produced by spray drying aqueous solutions of PTH asdescribed in A. above using a Buchi 190 mini spray dryer (BuchiLabortechnik AG, Meierseggstrasse, Switzerland) equipped with acustomized nozzle (Platz, R., et al., Inhale Therapeutic Systems'International Patent Publication No. WO 97/41833, Nov. 13, 1997) andcyclone. High collection efficiencies (yields), usually between about50-80%, were attained.

TABLE 6 PTH Dry Powder Compositions Emitted Dose, % Com- mean MMAD LotNo. position n = 10 RSD, % (μm) FPD R97190 30% PTH 62 4 — — 70% mannitol30% PTH 66 9 — — 70% raffinose R97191 75% PTH 51 3 — — 25% mannitol 30%PTH 78 — 2.43 0.58 70% leu 30% PTH 83 — 2.63 0.45 70% tri- leu

In looking at the results in Table 6 (and in other tables as well), itcan be seen that the addition of trileucine is effective tosignificantly improve the aerosol performance of the resulting powder.The aerosol performance of a PTH dry powder, as indicated by its EDvalue, was unexpectedly increased from 51-62% to 83% by the addition oftri-leucine to the formulation. These data illustrate a tremendousimprovement in emitted dose, achieved simply by addition of theexemplary surface active tripeptide, tri-leucine to the formulation.Surprisingly, even upon correcting on a mole-to-mole basis for thenumber of leucine amino acids contained in trileucine (3 moles leu permole of trileucine), trileucine is more effective than leucine, on a perweight basis, at increasing the dispersivity of dry powder compositionsfor delivery to the lung.

Example 3 Aerosol Properties of Albuterol-Trileucine Dry Powders

Dry powders containing the small molecule, albuterol, were prepared toexamine the effects of trileucine on the dispersivity/aerosol propertiesof dry powders containing a non-proteinaceous active agent.

A. Solution Formulation Preparation

Formulation solutions were prepared at a total solids content of 1%(w/v). For low solids-containing solutions, raffinose was added to bringthe total solids content to the above value. Table 7 lists thecompositions of all pre-spray dried solutions.

37. Powder Processing: Spray Drying

Powders were produced by spray drying aqueous solutions of albuterol,surface active di- or tri-peptide, and/or other excipient(s) using aBuchi 190 mini spray dryer (Buchi Labortechnik AG, Meierseggstrasse,Switzerland) as described in Example 2 above. Characteristics of theresultant powders are provided in Tables 7 and 8 below.

TABLE 7 Albuterol Dry Powders Formulation Emitted Dose, % Tg, ° C. 2%albuterol 31 102.2 98% raffinose 2% albuterol 31 88.57 5% leucineraffinose 2% albuterol 34 93.1 20% leucine raffinose 2% albuterol 7496.6 60% leucine raffinose 2% albuterol 62 85.3 5% trileucine raffinose2% albuterol 78 95.9 20% trileucine raffinose 2% albuterol 82 88.6 60%trileucine raffinose

TABLE 8 Additional Aerosol Properties of Albuterol Dry PowdersFormulation FPD MMAD, microns 2% albuterol 0.56 2.43 60% leucineraffinose 2% albuterol 0.59 2.43 20% tri-leucine raffinose

As can be seen from the results provided above, the addition oftrileucine increased the emitted dose of albuterol dry powders fromabout 30% to about 80%—an improvement in dispersivity of nearlythree-fold! Thus, the addition of a surface active di- or tri-peptide toan active agent dry powder can, by greatly improving the powder'sdispersivity, (i) reduce costly drug loses to the inhalation device,(ii) reduce the number of required inhalations per day by increasing theamount of aerosolized drug reaching the alveoli of a patient, (iii)reduce the amount of dry powder per unit dose, due to the highefficiency of aerosolization of dry powder, and (iv) increase the easeof manufacturing unit dosage forms of powdered drug, due to increasedflowability of powder.

Additionally, the addition of 60% by weight leucine was required toachieve the same level of dispersivity achieved by the addition of only20% by weight tri-leucine. Thus, tri-leucine is much more effective thanleucine in improving the aerosol performance of dry powders. Moreover, amaximum in aerosol performance is typically achieved by the addition ofonly from about 5-25% (wt) trileucine; quantities greater than thattypically provide only incremental improvements in dispersivity.

The dispersibility-enhancing effects of tri-leucine, and other surfaceactive di- and tri-peptides, appear to be general, and extend to notonly protein powders, but to powdered formulations of a wide variety ofactive agents (e.g., small molecules, hormones, antibiotics, and thelike), as illustrated by the Examples provided herein.

Example 4 Aerosol Properties of Salmon Calcitonin-Trileucine Dry Powders

The effects of trileucine on the aerosol performance of dry powderscontaining salmon calcitonin, a hormone with a molecular weight ofapproximately 4500 daltons, were examined.

Although salmon calcitonin is a highly surface active protein,spray-dried powders containing 5% (wt) salmon calcitonin and 95% (wt)raffinose exhibited relatively low emitted dose values (of approximately50%). In efforts to further explore the broad applicability of addingsurface active di- and tri-peptides to powder formulations to increasetheir dispersivity, tri-leucine was added to salmoncalcitonin-containing formulations to examine its impact on theresulting powders. The ability of tri-leucine to improve thedispersibility of salmon calcitonin containing dry powders was comparedto the amino acid, leucine.

Powders having the compositions indicated below were prepared asdescribed in Examples 2 and 3 above.

TABLE 9 S. Calcitonin Dry Powders Formulation Emitted Dose FPD Tg, ° C.5% s. Calcitonin 48 0.30 89.9 95% raffinose 5% s. Calcitonin 47 0.3189.3 5% leucine raffinose 5% s. Calcitonin 50 0.28 82.9 20% leucineraffinose 5% s. Calcitonin 48 0.29 82.3 40% leucine raffinose 5% s.Calcitonin 53 0.22 80.5 60% leucine raffinose 5% s. Calcitonin 64 0.2974.5 80% leucine raffinose 5% s. Calcitonin 58 0.46 89 5% tri-leucineraffinose 5% s. Calcitonin 72 0.50 91.1 20% tri-leucine raffinose 5% s.Calcitonin 76 0.46 83.4 40% tri-leucine raffinose 5% s. Calcitonin 840.49 94.3 60% tri-leucine raffinose 5% s. Calcitonin 86 0.49 115.2 80%tri-leucine raffinose

Representative mass median aerodynamic diameters were determined for twoof the formulations.

TABLE 10 Mass Median Aerodynamic Diameters of Calcitonin PowdersFormulation MMAD 5% s. Calcitonin 3.39 20% leucine raffinose 5% s.Calcitonin 2.87 20% tri-leucine raffinose

From the above data, it can be seen that tri-leucine can be used toimprove the aerosol properties of dry powder formulations of a widerange of active agents/medicaments for aerosolized delivery to the lung.

Trileucine provided nearly a 100% improvement in the emitted dose valueof a control powder containing salmon calcitonin and raffinose, nearlydoubling its ED value from 48% to 86%. Moreover, tri-leucine was moreeffective in enhancing powder dispersibility than leucine. While arepresentative formulation containing 80% by weight leucine exhibited anED value of 64%, formulations containing 60-80% tri-leucine possessed EDvalues from 84-86%, further indicating the superiority of tri-leucine insignificantly enhancing the aerosol performance of dry powders.

Example 5 Aerosol Properties of Antibiotic-Trileucine Dry Powders

The ability of tri-leucine to improve the dispersibility ofantibiotic-containing dry powders was explored.

A. Antibiotic Control Powders Absent Trileucine

Ciprofloxacin Powders. Aqueous solutions containing the componentspresented in Table 9 were prepared at a total solids content of 1%(w/v). The pH of each solution was determined, and solutions were thenspray-dried as described in Example 2 to prepare dry powders.

TABLE 11 Quantitative Composition Prior to Moisture MMAD Emitted BatchNumber Spray Drying¹ Content (μm) Dose (1) 1326-16 Ciprofloxacinhydrochloride 1136 mg 1.4% 2.8 42% DI water  113 ml (RSD = 8) solidproduct:  100% cipro (2) 1326-29 Ciprofloxacin hydrochloride 2047 mg3.2% 4.5 51% DI water  200 ml (RSD = 7) Sodium hydroxide QS to pH = 12solid product:  100% cipro (3) 1300-MG-7 Ciprofloxacin hydrochloride1995 mg 1.2% 2.9 33% Methanol  100 ml (RSD = 13) DI water  100 ml solidproduct:  100% cipro

Gentilmicin, Netilmicin Powders.

Dry powder compositions containing gentamicin or netilmicin wereprepared by mixing gentamicin sulfate or netilmycin sulfate andexcipient(s) (if used) with a liquid medium to form a solution. The pHof the solution was adjusted as appropriate to facilitate solubilizationand/or stabilization of the components in the solution. Quantitativeformulations are identified in Table 12 below. The solutions were thenspray-dried as described in Example 2 above to yield dry powders. Forformulations that utilized organic solvents, a modified Buchi 190 MiniSpray Dryer was used that was supplied with nitrogen as the gas sourceand equipped with an oxygen sensor and other safety equipment tominimize the possibility of explosion.

TABLE 12 Gentamicin/Netilmicin Dry Powders Batch Moisture MMAD NumberQuantitative Composition Content (μm) Emitted Dose 1326-31 Gentamicinsulfate 2076 mg 4.1%¹ 3.0 37% (RSD³ = 6) DI water  200 ml Hydrochloricacid QS to pH = 5 1326-32 Gentamicin sulfate 2053 mg 1.1%¹ 2.4 40% (RSD= 14) DI water  200 ml Sodium hydroxide QS to pH = 10 1300-MG-11Gentamicin sulfate 2012 mg 4.8%² 3.0 45% (RSD = 10) Ethanol  40 ml DIwater  160 ml 1300-MG-9 Netilmicin Sulfate 1626 mg 4.2% 3.2 47% (RSD =8) DI water  163 ml 1300-MG-14 Netilmicin Sulfate 1512 mg 5.1% 2.9 39%(RSD = 7) Ethanol  30 ml DI water  120 ml ¹Determined with Karl-Fischerreagent titrimetric method ²Determined with thermogravimetric analysis³Relative Standard Deviation

B. Trileucine-Containing Antibiotic Powders

Aqueous solutions (100 ml total volume) containing antibiotic andtri-leucine at a total solids content of 1% were prepared and the pH ofthe solutions adjusted to pH 4. The resulting solutions were thenspray-dried to produce powders having the relative amounts of antibioticand tri-leucine indicated in Table 13 below

TABLE 13 Antibiotic-Trileucine Dry Powders Formulation Yield, % MMAD, μmFPD (<3.3 μm) ED, % 95% Cipro 64.2 2.43 0.57 77.7 5% Leu-3 75% CiproN.A. 2.65 N.A. 83.0 25% Leu-3 45% Cipro 55.0 2.62 0.48 70.7 55% Leu-395% Gent. 61.4 2.15 0.66 75.7 5% Leu-3 75% Gent. 52.0 2.25 0.66 93.9 25%Leu-3 55% Gent. 54.2 2.51 0.51 87.3 45% Leu-3 95% Netil. 62.0 2.08 0.5882.4 5% Leu-3 75% Netil. 50.0 2.14 0.66 91.3 25% Leu-3 55% Netil. 40.02.73 0.49 90.4 45% Leu-3

As can be seen from the results in Table 13, the addition of tri-leucinewas effective to notably enhance the dispersibility of powders preparedfrom three different antibiotic compounds from two different antibioticclasses, ciprofloxacin (a quinolone), gentamicin and netilmicin(aminoglycosides). The ED values for ciprofloxacin powders increasedfrom values ranging from 33-51% to values ranging from 71-83%. Similarbeneficial results were observed for gentamicin powders, whose ED valueswere improved from 37-45% to 76-94% by addition of tri-leucine, and fornetilmicin, whose ED values improved from 39-47% to 82-91%. The optimalrelative amount of tri-leucine was determined for each of the threeantibiotic powders and determined to be approximately 25%, i.e., optimalED values were observed for powders containing 25% by weight tri-leucinerelative to antibiotic.

Example 6 Aerosol Properties of Powders Containing Interferon-β inCombination with Trileucine

The broad applicability of the use of surface active di- andtri-peptides for increasing powder dispersivity was further explored ininterferon-β powders. Interferon-β (a type I interferon) is a cytokinewith antiviral, antiproliferative, and immunomodulatory activity.

Powders containing interferon-β and optionally tri-leucine and/or otherexcipients (hydroxyethylstarch, HES and raffinose) were prepared asdescribed above. The solids content of the pre-dried solutions was 1%,with the exception of Lot No. RB27, which possessed a solids content of0.5%. The composition of the final powders is given in Table 14 below.

TABLE 14 Interferon-β Powders Containing Tri-leucine ED, % mean MMAD,Yield, % <5 Lot # Comp. (n = 10) RSD, % μm FPD, % % μm, % RB19 10% IFN-β81 7 3.2 48 56 79 45% Leu-3 45% HES RB21 10% IFN-β 80 6 2.9 46 61 85 45%Leu-3 45% Raff. RB24 10% IFN-β — — — —  9* — 90% Leu-3 RB27 10% IFN-β 744 2.9 49 40 81 67.5% Leu- 3 22.5% Raff RB29 10% IFN-β 79 5 3.2 41 50 8345% Leu-3 45% HES RB36 10% IFN-β 87 3 — — 61 — 22.5% Leu- 3 67.5% Raff.99320 10% IFN-β 64 — — — — — 90% Raff. *No tests performed due to lowyield.

As with the other active-agent containing powders, the addition oftri-leucine to powders composed of interferon-β served to increase thedispersivity and overall aerosol properties of the resulting powder.Although the improvement was not as striking in some of the previousexamples, addition of tri-leucine enhanced the ED values of aninterferon-β powder from 64% to 74-87%. As in the previous example, itappears that an optimal amount of tri-leucine is around about 22-25% byweight for the IFN-β powder.

Example 7 Factor IX Dry Powders

Powders containing factor IX, a 55,000 dalton glycoprotein with amodular domain structure and numerous posttranslational modifications,useful in the treatment of hemophilia B, and trileucine and/or otherexcipient(s), were prepared to further explore thedispersivity-enhancing effects of tri-leucine and other surface activedi- and tri-peptides on different medicaments.

Powders containing Factor IX, both with and without leucine or aleucine-containing dimer or trimer, were prepared as describedpreviously. The solids content of the pre-spray-dried solution was 1% byweight (w/v). Yields of the spray dried powders ranged from 40 to 60%.The formulations of the dried powders are provided in Table 15 below.

TABLE 15 Factor IX Powders Emitted Dose Formulation (RSD) MMAD 93%Factor IX/7% 57 — NaCitrate (5%) 37% Factor IX/3% Na 78 2.9 Citrate/60%Leucine (3%) 56% Factor IX/4% Na 89 2.7 Citrate/40% Trileucine (5%)

The results in Table 15 further support the effectiveness of tri-leucineat significantly improving the dispersibility of dry powdercompositions, irrespective of the active agent contained in thecomposition. Moreover, as in the previous examples, tri-leucine isbetter than leucine in significantly improving the dispersibility of thecomposition (from an ED of 57% to 89%), and can achieve such enhancementwhen used in smaller quantities than leucine.

Example 8 Stability Studies

The chemical and physical stability of packaged PTH powders underaccelerated stability conditions were evaluated on the basis of thechange in protein concentration and aerosol properties measured betweeninitial and 3-month time points. PTH-trileucine and PTH-leucine powderswere prepared as in Example 2 above.

Powders were hand-filled in blister packs (BPs). The blister packs wereplaced in petri dishes (20-60 BPs/dish).

TABLE 16 Accelerated stability study at 40° C./Ambient Relative HumidityPackaged Fine Storage Condition Particle no 2^(nd) wrap % Emitted Dose %Wt. Formulation ID no dessicant % Purity Dose (FPD) MMAD ChangeComposition 40° C./ambient RH (by area) (rsd) <3.3 μm (μm) (TGA) R99484initial 97.0 79.6 (3) 0.58 2.5 1.4 30% PTH/  4 weeks n/a 74.9 (5) n/an/a 1.7 70% Leucine  6 weeks n/a 75.2 (6) n/a n/a n/a  8 weeks 95.2*78.8 (6) 0.55 2.4 2.2 12 weeks n/a 78.6 (3) n/a n/a TBD R99485 initial97.1 79.4 0.45 2.9 2.6 30% PTH/70%  4 weeks n/a 75.8 n/a n/a 2.4tri-leucine  6 weeks n/a 81.6 n/a n/a n/a  8 weeks 94.8 81.6 0.44 2.92.4 12 weeks n/a out of BP n/a out of out of BP BP *the chemicalstability of the 8 weeks, 40° C./ambient RH sample is similar to thestability of the 6 months, 40° C./dry sample (foiled wrapped w/desiccants) of a 30% PTH/70% mannitol formulation.

In looking at the results in Table 16, it can be seen that thetrileucine-containing formulation is both chemically and physicallystable upon storage, even at temperatures increased over ambient.Specifically, the 30% PTH/70% trileucine powder exhibited minimaldegradation of protein over the timecourse of 3 months, while theaerosol performance of the powder remained essentially unchanged.

Example 9 Electron Spectroscopy of Chemical Analysis (ESCA) of PowderFormulations

ESCA analysis was carried out on certain powder formulations toinvestigate the surface enrichment of di-leucyl di- or tripeptide in theparticles. The relative concentrations of powder components in the bulkpowder is provided in the column, “Formulation”; the concentration ofeach component on the surface of the particles, as determined by ESCA,is provided in the column, “ESCA result”.

TABLE 17 SCal/Raffinose/Leu formulations pH7 Lot No. Formulation (% w/w)ESCA result (% w/w) R99282 sCal 5 53 pH7 Leucine 0 — Raffinose 95 47R99283 sCal 5 11 pH7 Leucine 5 52 Raffinose 90 38 R99284 sCal 5 39 pH7Leucine 20 28 Raffinose 75 33 R99286 Scal 5 26 pH7 Leucine 80 64Raffinose 15  9

TABLE 18 Leucyl-Peptide/Raffinose formulations Lot No. Formulation (%w/w) ESCA result (% w/w) R99337 Leucine-2 5 28.7 pH7 Raffinose 95 71.3R99338 Leucine-2 20 44.1 pH7 Raffinose 80 55.9 R99339 Leucine-2 60 94.9pH7 Raffinose 40 5.1 R99340 Leucine-3 20 97.1 pH7 Raffinose 80 2.9R99342 Alanine-3 20 41.3 pH7 Raffinose 80 58.7

TABLE 19 SCal/Raffinose/Leu-3 formulations pH4 Lot No. Formulation (%w/w) ESCA result (% w/w) R99435 Scal 5 36.6 pH4 Leucine-3 0 0 Raffinose95 63.4 Scal 5 17.9 pH4 Leucine-3 5 7.0 Raffinose 90 75.1 R99437 Scal 546.4 pH4 Leucine-3 20 24.2 Raffinose 75 29.4 R99438 Scal 5 22.7 pH4Leucine-3 40 74.8 Raffinose 55 2.5 R99439 Scal 5 16.4 pH4 Leucine-3 6081.6 Raflinose 35 2.0

The above results indicate that powders containing a surface activematerial are enriched at the surface in concentration of surface activematerial. Surface enrichment of di- or trileucine is observed for boththe non-active agent containing powders in Table 18 and for the s.calcitonin powders in Table 19.

Although the ESCA results for the calcitonin powders are subject to somevariability (this is due to the difficulty of separating out surfaceconcentration contributions by components having the same atom withintheir structures i.e., calcitonin vs. trileucine), the overall trendobserved supports the finding of powders in which the surfaceconcentration of the di-leucyl di- or tripeptide is greater than that inthe bulk powder.

What is claimed is:
 1. A composition comprising an active agent and adi- or tripeptide selected from the group consisting of dileucine,leu-leu-gly, leu-leu-ala, leu-leu-val, leu-leu-leu, leu-leu-ile,leu-leu-met, leu-leu-pro, leu-leu-phe, leu-leu-trp, leu-leu-ser,leu-leu-thr, leu-leu-cys, leu-leu-tyr, leu-leu-asp, leu-leu-glu,leu-leu-lys, leu-leu-arg, leu-leu-his, leu-leu-nor, leu-gly-leu,leu-ala-leu, leu-val-leu, leu-ile-leu, leu-met-leu, leu-pro-leu,leu-phe-leu, leu-trp-leu, leu-ser-leu, leu-thr-leu, leu-cys-leu,leu-tyr-leu, leu-asp-leu, leu-glu-leu, leu-lys-leu, leu-arg-leu,leu-nor-leu, and combinations thereof.
 2. The composition of claim 1, inaerosolized form.
 3. The composition of claim 1, in liquid form.
 4. Thecomposition of claim 3, wherein the liquid is an aqueous solution. 5.The composition of claim 3, wherein the liquid is a suspension.
 6. Thecomposition of claim 3, further comprising a propellant.
 7. Thecomposition of claim 6, wherein the propellant is selected from thegroup consisting of a chlorofluorocarbon, a fluorocarbon, andcombinations thereof.
 8. The composition of claim 7, wherein thepropellant comprises a chlorofluorocarbon or fluorocarbon.
 9. Thecomposition of claim 3, in aerosolized form.
 10. The composition ofclaim 3, wherein the composition is suitable for delivery to the lung ordeep lung by inhalation.
 11. The composition of claim 10, wherein thecomposition is suitable for delivery via nebulization.
 12. Thecomposition of claim 3, wherein the liquid comprises a solvent.
 13. Thecomposition of claim 12, wherein the solvent is selected from the groupconsisting of water, ethanol, saline, and combinations thereof.
 14. Thecomposition of claim 1, wherein the di- or tripeptide is present in thecomposition from about 1% by weight to about 99% by weight.
 15. Thecomposition of claim 14, wherein the di- or tripeptide is present in thecomposition from about 2% by weight to about 75% by weight.
 16. Thecomposition of claim 15, wherein the di- or tripeptide is present in thecomposition from about 5% by weight to about 50% by weight.
 17. Thecomposition of claim 1, further comprising a pharmaceutically acceptableexcipient or carrier.
 18. The composition of claim 17, wherein theexcipient is selected from the group consisting of carbohydrates, aminoacids, peptides, proteins, organic acid salts, and polymers.
 19. Thecomposition of claim 1, comprising a dipeptide.
 20. The composition ofclaim 19, wherein the dipeptide is dileucine.
 21. The composition ofclaim 1, comprising a tripeptide.
 22. The composition of claim 21,wherein the tripeptide is selected from the group consisting ofleu-leu-leu, leu-leu-val, leu-leu-ile, leu-leu-trp, leu-leu-ala,leu-leu-met, leu-leu-phe, leu-leu-tyr, leu-leu-pro, leu-val-leu,leu-ile-leu, leu-trp-leu, leu-ala-leu, leu-met-leu, leu-phe-leu,leu-tyr-leu, leu-pro-leu, and combinations thereof.
 23. The compositionof claim 22, wherein the tripeptide is trileucine.
 24. The compositionof claim 1, wherein said active agent is selected from the groupconsisting of insulin, cyclosporin, parathyroid hormone, folliclestimulating hormone, VLA-4 inhibitors, interleukin-4R, thrombopoietin,c-peptide, amylin, pro-insulin, interleukin-1, interleukin-2,alpha-1-antitrypsin, budesonide, human growth hormone, growh hormonereleasing hormone, interferon alpha, interferon beta, growth colonystimulating factor, keratinocyte growth factor, glial growth factor,tumor necrosis factor, leutinizing hormone releasing hormone,calcitonin, low molecular weight heparin, somatostatin, respiratorysyncytial virus antibody, erythropoietin, Factor VIII, Factor IX,ceredase, cerezyme and analogues, agonists and antagonists thereof. 25.The composition of claim 1, in a dry powder form.
 26. The composition ofclaim 25, wherein the dry powder is a spray-dried powder.
 27. A methodfor treating a mammalian subject suffering from a condition that isresponsive to an active agent comprising administering to the subject acomposition comprised of a therapeutically effective amount of theactive agent and a di- or tripeptide selected from the group consistingof dileucine, leu-leu-gly, leu-leu-ala, leu-leu-val, leu-leu-leu,leu-leu-ile, leu-leu-met, leu-leu-pro, leu-leu-phe, leu-leu-trp,leu-leu-ser, leu-leu-thr, leu-leu-cys, leu-leu-tyr, leu-leu-asp,leu-leu-glu, leu-leu-lys, leu-leu-arg, leu-leu-his, leu-leu-nor,leu-gly-leu, leu-ala-leu, leu-val-leu, leu-ile-leu, leu-met-leu,leu-pro-leu, leu-phe-leu, leu-trp-leu, leu-ser-leu, leu-thr-leu,leu-cys-leu, leu-tyr-leu, leu-asp-leu, leu-glu-leu, leu-lys-leu,leu-arg-leu, leu-nor-leu, and combinations thereof.